Journal: The Journal of Clinical Investigation

Article Title: GIGYF1 disruption associates with autism and impaired IGF-1R signaling

doi: 10.1172/JCI159806

Figure Lengend Snippet: ( A ) Coimmunoprecipitation assay for GIGYF1 and IGF-1R in HEK293T cells. ( B ) Double immunofluorescence of GIGYF1 and IGF-1R in HeLa cells. Scale bars: 10 μm. Inset scale bars: 5 μm. ( C ) Immunoblots of the whole cell lysate showing levels of pIGF-1R, IGF-1R, pERK1/2, ERK1/2, pAkt, and Akt at different duration of IGF-1 stimulation. Statistical data were analyzed using 2-way ANOVA. ( D ) Immunoblots of the whole-cell lysates showing levels of pERK1/2, ERK1/2 in HEK293T GIGYF1 KO cells expressing mock empty vector (pCAGGS-IRES-GFP) or HA-GIGYF1. The relative levels of pERK1/2 to ERK1/2 were quantified by densitometry and analyzed using 2-way ANOVA. ( E ) Immunoblot of pERK1/2 and ERK1/2 in the whole-cell lysates at 7 minutes of IGF-1 stimulation. Statistic data were analyzed using 1-way ANOVA. ( F ) Immunoblots of biotin-labelled IGF-1R, total IGF-1R and TMEM98. Total IGF-1R-α levels of unbiotinylated cells were determined. The protein levels of surface IGF-1R, surface IGF-1R/total IGF-1R, and total IGF-1R were quantified by densitometry from 3 biological replicates. Statistical data were analyzed using 2-tailed Student’s t tests. ( G ) Immunoblots of biotin-labeled IGF-1R and total IGF-1R at different conditions in a surface biotinylation recycling assay. GIGYF1 KO HEK293T cells and control HEK293T cells (lanes 2–8) were surface labeled with sulfo-NHS-S-S-biotin. Cells (lanes 3–8) were incubated to endocytosis. The remaining surface biotin was cleaved with glutathione cleavage buffer (lanes 2–8). Cells were incubated for a second time to recycle (lanes 4–5 and 7–8), then the surface biotin was stripped for a second time (lanes 3–4 and 6–7). Lanes 5 and 8 were incubated to recycle without a second cleavage. The stage of biotinylation recycling assay for each lysate is indicated with + and –. ( H ) Working model of GIGYF1 regulation of IGF-1R/ERK pathway. All data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. See complete unedited blots in the supplemental material.

Article Snippet: The following primary antibodies were used: rabbit HA-tag (1:1,000, CST, 3724s), rabbit Flag-tag (1:1,000, CST, 14793s), rabbit Clathrin Heavy Chain (1:50, CST, 4796s), rabbit Caveolin-1 (1:200, CST, 3267s), rabbit EEA1 (1:200, CST, 3288s), mouse Rab5A (1:400, CST, 46449s), rabbit Rab4 (1:200, CST, 2167s), rabbit Rab11 (1:50, CST, 5589s), rabbit Rab7 (1:50, CST, 9367s), rabbit Lamp1 (1:100, CST, 9091s), rabbit phospho-IGF-1R-β (Tyr1131)/insulin receptor β (Tyr1146) (1:1,000, CST, 3021s), rabbit IGF-1R-β (1:1,000, CST, 9750s), rabbit phospho-Akt (Ser473) (1:1,000, CST, 4060s), rabbit Akt (1:1,000, CST, 9272s), phospho-p44/42 MAPK (Thr202/Tyr204) (1:1,000, CST, 4370s), rabbit p44/42 MAPK (1:1,000, CST, 9102s), rabbit p27 Kip1 (1:1,000, CST, 3686s), rabbit Cyclin D1 (1:1,000, CST, 55506s), rabbit Tbr1 (1:400, CST, 49661s), rabbit Ki67 (1:400, CST, 9129s), rabbit Grb10 (1:1,000, Santa Cruz, sc-74509), mouse IGF-1R-α (1:1,000, Santa Cruz, sc-81464), mouse Brn2 (1:100, Santa Cruz, sc-393324), rabbit Satb2 (1:500, Abcam, ab92446), rabbit Tbr2 (1:100, Abcam, ab23345), rat BrdU (1:500, Abcam, ab6326), rabbit Ctip2 (1:100, Abcam, ab18465), rabbit 488 (1:500, Thermo Fisher Scientific, SA5-10018), rabbit Cy3 (1:500, Thermo Fisher Scientific, A10522), chicken GFP (1:500, Aves Labs, GFP-1020), rabbit Pax6 (1:100, BioLegend, PRB-278P), rabbit GIGYF1 (1:1,000, Bethyl, A304-132A-M), mouse Ph3 (1:2,000, Millipore-Sigma, 07-424), and rabbit TMEM98 (1:1,000, Millipore-Sigma, HPA053385).

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Western Blot, Expressing, Plasmid Preparation, Labeling, Control, Incubation